Stimulation of RBL-2H3m1 mast cells through the IgE receptor with antigen, or through a G protein-coupled receptor with carbachol, leads to the rapid appearance of phosphothreonine in nonmuscle myosin heavy chain II-A (NMHC-IIA). We demonstrate that this results from phosphorylation of Thr-1940 by CaM kinase II and can be mimicked by increasing intracellular calcium with the calcium ionophore ionomycin. The phosphorylation site in rodent NMHC-IIA was localized to the carboxyl terminus of NMHC-IIA distal to the coiled-coil region and identified as Thr-1940 by site-directed mutagenesis. A fusion protein containing the NMHC-IIA C-terminus was phosphorylated by calcium/calmodulin-dependent protein kinase II (CaM kinase II) in vitro, while mutation of Thr-1940 to Ala eliminated phosphorylation. In contrast to rodents, in humans, Thr-1940 is replaced by Ala and human NMHC-IIA fusion protein was not phosphorylated by CaM kinase II unless Ala-1940 was mutated to Thr. In vitro phosphorylation of NMHC-IIA fusion protein was inhibited by the metastasis-associated calcium binding protein Mts1, which has been shown to bind to the C-terminus of NMHC-IIA. Co-transfection of Thr(Ala)1940 human NMHC-IIA and activated CaM kinase II in HeLa cells resulted in phosphorylation of the mutated NMHC, while wild type myosin was not phosphorylated. In RBL-2H3 cells, inhibition of CaM kinase II decreased Thr-1940 phosphorylation and inhibited release of the secretory granule marker hexosamindase in response to carbachol, but not to antigen. These data indicate a role for CaM kinase stimulation and resultant threonine phosphorylation of NMHC-IIA in RBL-2H3m1 cell activation.